Lead detection method and reggents utilizing aminolevulinic acid dehydratase and tertiary phosphines

Abstract

A method and kit for simplifying and improving the sensitivity and accuracy of a lead assay for a sample solution suspected of containing lead determines the extent of a reaction between a substrate and a disulfide enzyme in the presence of an activating reagent which contains a water-soluble tertiary phosphine reagent so as to increase the activity of the disulfide enzyme for reaction with the substrate. For a colorimetric determination of the enzyme activity a chromophore is formed upon reaction with a selected component of the sample solution in the presence of a colorimetric enhancing reagent. The colorimetric enhancing reagent contains a metal ion such as cupric ion or ferric ion which is soluble in the sample solution. The extent of the chromophore formation is then photometrically determined.

What is claimed is: 
     
       1. A method for detecting lead in a sample suspected of containing lead, the method comprising: (a) forming an aqueous solution from the sample such that any lead in the sample is present in said aqueous solution;   (b) contacting the aqueous solution with an aminolevulinic acid dehydratase enzyme in the presence of a water soluble tertiary phosphine;   (c) incubating the enzyme with aminolevulinic acid to form a product comprising porphobilinogen;   (d) stopping said enzyme incubation performed in step (c) after a predetermined time interval;   (e) detecting said porphobilinogen resulting from step (c); and   (f) correlating said porphobilinogen detected in step (e) with the presence of lead in the sample whereas porphobilinogen detected indicates the presence of lead in said sample.   
     
     
       2. The method of claim 1 wherein the water soluble tertiary phosphine reagent is tris(2-carboxyethyl) phosphine. 
     
     
       3. The method of claim 1 wherein the water soluble tertiary phosphine is selected from the group consisting of tributylphosphine, tris(carboxyphenyl)phosphine, and tris(hydroxymethyl)phosphine. 
     
     
       4. The method of claim 1 wherein the method further comprises forming a chromophore with said porphobilinogen in the presence of a colorimetric enhancing reagent, said colorimetric enhancing reagent comprising a cupric ion soluble in the aqueous solution. 
     
     
       5. The method of claim 4 wherein the colorimetric enhancing reagent is added directly to the aqueous solution after incubation of the aminolevulinic acid dehydratase and aminolevulinic acid in step (c). 
     
     
       6. The method of claim 1 wherein the method comprises acidifying the aqueous solution to isolate the lead from compounds which interfere with said method, said compounds being selected from the group consisting of proteins, endogenous d-aminolevulinic acid dehydratase, porphobilinogen and aminolevulinic acid, and neutralizing the aqueous solution before said enzyme incubation of step (c). 
     
     
       7. The method of claim 1 wherein the stopping step includes acidifying the aqueous solution and adding a coloring reagent to form a chromophore upon reaction with said porphobilinogen. 
     
     
       8. The method of claim 7 wherein the coloring reagent is selected from the group consisting of dimethylaminobenzaldehyde and dimethylaminocinnamaldehyde. 
     
     
       9. The method of claim 1 wherein the stopping step includes forming a chromophore upon reaction of a coloring reagent with the porphobilinogen in the presence of a colorimetric enhancing reagent, the colorimetric enhancing reagent comprising a cupric ion soluble in the aqueous solution; and wherein step (e) includes photometrically detecting said chromophore. 
     
     
       10. The method of claim 9 wherein the cupric ion is present in an amount of about 1 mM to about 500 mM. 
     
     
       11. A method for detecting lead in a sample suspected of containing lead, the method comprising: (a) forming an aqueous solution from the sample such that any lead in the sample is present in said aqueous solution;   (b) incubating in said aqueous solution, (i) an aminolevulinic acid dehydratase enzyme; and (ii) aminolevulinic acid, in the presence of a reducing agent, to form porphobilinogen;   (c) stopping said enzyme incubation of step (b) after a predetermined time interval;   (d) contacting the porphobilinogen formed in step (b) with a coloring reagent to form a chromophore, in the presence of a metal ion selected from the group consisting of cupric ion and ferric ion;   (e) photometrically detecting said chromophore resulting from step (d); and (f) correlating the chromophore detected in step (e) with the presence of lead in the sample whereas the presence of the chromophore detects lead in the sample.   
     
     
       12. The method of claim 11 wherein the method further includes acidifying the aqueous solution to isolate the lead in the solution from compounds which interfere with said method, said compounds being selected from the group consisting of proteins, endogenous d-aminolevulinic acid dehydratase, porphobilinogen and aminolevulinic acid, and neutralizing the aqueous solution before said enzyme incubation of step (b). 
     
     
       13. The method of claim 11 wherein step (c) includes acidifying the aqueous solution and adding a coloring reagent to the solution. 
     
     
       14. The method of claim 13 wherein the coloring reagent is selected from the group consisting of dimethylaminobenzaldehyde and dimethylaminocinnamaldehyde. 
     
     
       15. The method of claim 11 wherein said metal ion is cupric ion present in an amount of about 100 mM to about 500 mM. 
     
     
       16. The method of claim 11 wherein said metal ion is ferric ion present in an amount of about 10 mM to 100 mM. 
     
     
       17. The method of claim 11 wherein the reducing agent comprises dithiothreitol. 
     
     
       18. A lead assay reagent composition comprising (i) an aminolevulinic acid dehydratase enzyme, or its substrate aminolevulinic acid; and (ii) a water soluble tertiary phosphine. 
     
     
       19. The reagent composition of claim 18 wherein the water soluble tertiary phosphine is selected from the group consisting of tris(2-carboxyethyl)phosphine tributylphosphine, tris(carboxyphenyl)phosphine, and tris(hydroxymethyl)phosphine. 
     
     
       20. The reagent composition of claim 18 wherein the reagent composition further comprises a colorimetric enhancing reagent, said colorimetric enhancing reagent comprising a cupric ion soluble in the reagent composition. 
     
     
       21. The reagent composition of claim 20 wherein the cupric ion is present in an amount of about 100 mM to about 500 mM. 
     
     
       22. A lead assay reagent composition comprising (i) a metal ion selected from the group consisting of cupric ion and ferric ion, and (ii) a coloring reagent capable of forming a photometrically detectable chromophore upon reaction with porphobilinogen. 
     
     
       23. The reagent composition of claim 22 wherein the coloring reagent is selected from the group consisting of dimethalaminobenzaldehyde and dimethalaminocinnemaldehyde. 
     
     
       24. The reagent composition of claim 22 wherein the reagent composition further comprises a trichloroacetic acid stop reagent for stopping the reaction between said enzyme and said aminolevulinic acid. 
     
     
       25. The reagent composition of claim 22 wherein the reagent is cupric ion present in an amount of about 100 mM to about 500 mM. 
     
     
       26. The reagent composition of claim 22 wherein the reagent is ferric ion present in an amount of about 10 mM to 100 mM. 
     
     
       27. A kit for performing an assay for lead on a sample suspected of containing lead, the kit comprising (i) a first container comprising an aminolevulinic acid dehydratase enzyme; (ii) a second container, separate from said first container, comprising aminolevulinic acid, wherein said first or said second container further comprises a water soluble tertiary phosphine. 
     
     
       28. The kit of claim 27 wherein the kit further includes a container comprising (i) a stop reagent which stops the reaction between the aminolevulinic acid and aminolevulinic acid dehydratase enzyme and (ii) a coloring reagent which forms a chromophore upon reaction with porphobilinogen. 
     
     
       29. The kit of claim 28 wherein the stop reagent further comprises a colorimetric enhancing reagent, said colorimetric enhancing reagent containing a cupric ion soluble in the stop reagent. 
     
     
       30. The kit of claim 27 wherein said water soluble tertiary phosphine is tris(2-carboxyethyl) phosphine. 
     
     
       31. The kit of claim 27 wherein the water soluble tertiary phosphine is selected from the group consisting of tributylphosphine, tris(carboxyphenyl)phosphine, and tris(hydroxymethyl)phosphine. 
     
     
       32. A kit for performing an assay on a sample suspected of containing lead, the kit comprising: a container comprising a substrate aminolevulinic acid;   a container comprising an aminolevulinic acid dehydratase enzyme;   a container comprising a trichloroacetic acid stop reagent effective for stopping the reaction between the substrate and the enzyme, a coloring reagent for forming a chromophore upon reaction with porphobilinogen, and a colorimetric enhancing reagent for improving the sensitivity and accuracy of photometrically determining the extent of the chromophore formation, said colorimetric enhancing reagent comprising a metal ion soluble in the stop reagent selected from the group consisting of cupric ion and ferric ion; and   a reducing reagent present in either the substrate container, or the enzyme container in an amount effective to increase reaction of the enzyme with said substrate.   
     
     
       33. The kit of claim 32 wherein the colorimetric enhancing reagent is cupric ion present in an amount of about 100 mM to about 500 mM. 
     
     
       34. The kit of claim 32 wherein the colorimetric enhancing reagent is ferric ion present in an amount of about 10 mM to 100 mM. 
     
     
       35. The kit of claim 32 wherein the reducing agent comprises dithiothreitol. 
     
     
       36. The kit of claim 32 wherein the coloring reagent is selected from the group consisting of dimethylaminobenzaldehyde and dimethylaminocinnamaldehyde.